Romanov V, Hausinger R P
Institute for Biochemistry and Physiology of Microorganisms, Russian Academy of Science, Pushchino, Moscow Region.
J Bacteriol. 1994 Jun;176(11):3368-74. doi: 10.1128/jb.176.11.3368-3374.1994.
Cell extracts of Pseudomonas aeruginosa 142, which was previously isolated from a polychlorinated biphenyl-degrading consortium, were shown to degrade 2,4-dichlorobenzoate, 2-chlorobenzoate, and a variety of other substituted ortho-halobenzoates by a reaction that requires oxygen, NADH, Fe(II), and flavin adenine dinucleotide. By using extracts that were chromatographically depleted of chlorocatechol and catechol 1,2-dioxygenase activities, products of the initial reaction with 2,4- or 2,5-dichlorobenzoate and 2-chlorobenzoate were identified by mass spectrometry as 4-chlorocatechol and catechol. In contrast to the well-characterized benzoate dioxygenases or the recently described 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS (S. Fetzner, R. Müller, and F. Lingens, J. Bacteriol. 174:279-290, 1992) that possess two protein components, the P. aeruginosa enzyme was resolved by ion-exchange chromatography into three components, each of which is required for activity. To verify the distinct nature of this enzyme, we purified, characterized, and identified one component as a ferredoxin (M(r), approximately 13,000) containing a single [2Fe-2S] Rieske-type cluster (electron paramagnetic resonance spectroscopic values of gx = 1.82, gy = 1.905, and gz = 2.02 in the reduced state) that is related in sequence to ferredoxins found in the naphthalene and biphenyl three-component dioxygenase systems. By analogy to these enzymes, we propose that the P. aeruginosa ferredoxin serves as an electron carrier between an NADH-dependent ferredoxin reductase and the terminal component of the ortho-halobenzoate 1,2-dioxygenase. The broad specificity and high regiospecificity of the enzyme make it a promising candidate for use in the degradation of mixtures of chlorobenzoates.
铜绿假单胞菌142的细胞提取物先前是从一个多氯联苯降解菌群中分离出来的，该提取物可通过一种需要氧气、NADH、Fe(II)和黄素腺嘌呤二核苷酸的反应来降解2,4 -二氯苯甲酸、2 -氯苯甲酸以及多种其他取代的邻卤苯甲酸。通过使用经色谱法去除了氯儿茶酚和儿茶酚1,2 -双加氧酶活性的提取物，用质谱法鉴定出与2,4 -或2,5 -二氯苯甲酸和2 -氯苯甲酸初始反应的产物为4 -氯儿茶酚和儿茶酚。与已充分表征的苯甲酸双加氧酶或最近描述的来自洋葱伯克霍尔德菌2CBS的2 -卤苯甲酸1,2 -双加氧酶（S. Fetzner、R. Müller和F. Lingens，《细菌学杂志》174:279 - 290，1992年）不同，后者具有两个蛋白质组分，而铜绿假单胞菌的这种酶通过离子交换色谱法可分离为三个组分，每个组分对于活性都是必需的。为了验证这种酶的独特性质，我们对其中一个组分进行了纯化、表征并鉴定为一种铁氧还蛋白（相对分子质量约为13,000），它含有一个单一的[2Fe - 2S] Rieske型簇（还原态下电子顺磁共振光谱值为gx = 1.82、gy = 1.905和gz = 2.02），其序列与萘和联苯三组分双加氧酶系统中发现的铁氧还蛋白相关。通过与这些酶类比，我们提出铜绿假单胞菌铁氧还蛋白在依赖NADH的铁氧还蛋白还原酶和邻卤苯甲酸1,2 -双加氧酶的末端组分之间充当电子载体。该酶广泛的底物特异性和高区域特异性使其成为用于降解氯苯甲酸混合物的有前景的候选物。
